Lipases are enzymes that catalyse the hydrolysis of ester bonds in lipid substrates, leading to the release of fatty acids. Lipases are used in the dairy applications for flavour generation, most importantly in cheese. Traditionally, ruminant lipase preparations are used, derived from goat, kid goat, calf or lamb. These are derived from pregastric tissues from these ruminants and these lipase preparations are also referred to as pregastric esterases. Commercial preparations are in the market, such as the Piccantase® C, L, KG and K (DSM Food Specialties, The Netherlands). These lipases are used in the preparation of a variety of Italian, Spanish, Greek and French cheeses. The development of a specific flavour profile in these types of cheeses during ripening is largely due to the action of lipases on milk fat. Lipases catalyse hydrolysis of milk fat with generation of free fatty acids. Said fatty acids may have short chains (C4-C6 fatty acids, such as containing 4 or 6 carbon atoms, i.e. butyric, caproic acid) and medium to long chain (C12-C18 fatty acids). Subsequently free fatty acids can take part in chemical reactions, e.g. the formation of flavour compounds such as acetoacetate, beta-keto acids, methyl ketons, esters and lactones. Conversion of fatty acids in flavor components can be catalysed by the enzymes originating from the microbial population in cheese.
It is known that the type of free fatty acids released by lipases in cheese can be influenced by the type of lipases used. For example lipases that primarily release short chain fatty acids (e.g. C4 and C6 containing fatty acids) lead to the development of a piquant, sharp, spicy, tangy flavour, while release of medium to long chain fatty acids can lead to a soapy taste. Lipases find increasing use in other dairy applications than cheese, such as Enzyme Modified Cheese (EMC; Wilkinson et al in Encyclopedia of Dairy Sciences, (2003; Fox et all eds, Academic Press) pp. 434-438) or the hydrolysis of butter fat and cream and their applications (Kilara in Enzyclopedia of Dairy Sciences, (2003; Fox et all eds, Academic Press) pp. 914-918).
Ruminant lipases are preferred over microbial lipases because of their specificity to release short chain fatty acids (C4-, C6-fatty acids) from milk fat. These compounds are either flavour compounds themselves or are converted into volatile esters with a particular flavour impact (Liu et al, Int. Dairy J. 2004, 14, 923-945). An interesting issue is the composition of ruminant lipases, which is the topic of several papers (e.g. Addis et al Int. dairy J. (2005) 15, 1271-1278; Richardson et al, J. Dairy Sci. (1967) 50, 1061-1065; Addid et al Int. Dairy J. (2005) 15, 563-569; Hamosh Nutrition (1990) 6, 421-428; Calvo et al (2004) J. Dairy Sci. 87, 1132-1142). The data presented lead to the conclusion that most ruminant enzymes are probably mixtures of 2 or more lipases, and that variations in composition occur leading to changes in performance in cheese flavour formation. This variation is a driver for the industry to look for alternative enzyme sources with improved consistence. The occurrence of animal diseases like scrapie and mad cows disease is another driver for industry to look for alternatives. Further support comes from the desire to have easy access to Kosher and Halal quality products. There is therefore a strong industrial desire for alternatives for animal derived lipases.
Patent application US2004/0001819 described the cloning and expression of kid pregastric esterase in the yeast Pichia pastoris. Although potentially interesting, the enzyme is poorly produced and in addition the free fatty acid release profile shifted to longer chain fatty acids, as compared to the original kid goat esterase. These two aspects made this enzyme unattractive because of poor economics and lack of performance in application. A preferred alternative would be microbial lipases or (microbial) lipases recombinantly produced by micro-organisms. Several microbial lipases are in the market (for examples see e.g. Bjurlin et al, JAOCS (2001) 78, 153-160). The most important characteristic of microbial lipases for cheese application is their fatty acid release profile from milk fat, which should mimic as close as possible the animal derived lipases. Microbial lipases are, however, poor performers in this respect since they have a preference for the release of long chain (C12-C18) fatty acids relative to short chain fatty acids (C4, C6). This often leads to the formation of a soapy taste and not to the desired piquant flavour. Therefore, despite the fact that there is a considerable number of commercial microbial lipase preparations in the market there is still an industrial need for a non-animal derived lipase that can replace the animal derived lipases such as ruminant pregastric lipases.
An interesting issue is the composition of the ruminant lipases, also referred to as pregastric esterases. This item is addressed in several papers, and the data that are presented lead to the conclusion that the ruminant enzyme preparations most probably contain a mixture of two or more lipases/esterases. The composition of the ruminant preparations is important for their performance in cheese flavor formation. The mode of action, specificity and flavor effects of various ruminant preparations has been described in various papers.
Several pregastric esterases have been purified and/or cloned, e.g. from kid, goat, calf and lamb. They are similar with respect to physical properties and substrate preference.
In general they are glycosylated proteins with a molecular weight between 40-60 kD. The pH optima are in the neutral to slightly acidic region, which is well compatible with the pH found in cheese and EMC. All have a preference for the short chain fatty acids. From some species like lamb, different enzymes are isolated with slight differences in substrate preferences. This supports the view that pregastric esterase preparations may contain multiple lipases. The esterases preferentially release fatty acids from the sn1 or sn3 position from triacyl-glycerides. They are much less active on mono- and di-acylglycerides and phospholipids. This in contrast to many microbial lipases that are well able to degrade di- and mono-glycerides and often show activity on phospholipids as well.
The kid goat pregastric esterase has been cloned and heterogeneously expressed in the yeast Pichia pastoris. The expression levels are unclear, but the fatty acid release profile was reported. The recombinant enzyme shows a profile that deviates from the kid-derived preparation. The main difference is a lower release of short chain fatty acids for the recombinant enzyme. This finding could point to the multi-enzyme composition of the pregastric kid lipase. The calf pregastric lipase has been cloned and sequenced (Timmermans M Y, Teuchy H, Kupers L P. The cDNA sequence of bovine pregastric esterase. Gene (1994) 147, 259-262), but the efficient, commercial interesting, over-expression of the enzyme has not been described. The lamb pregastric lipase has not been cloned and sequenced.
Several other non-microbial lipases have been cloned, sequenced and characterized. These include several human lipases, rat lipases, pig hepatic lipase, and lipases from cat, dog, guinea pig, mouse, rabbit and tortoise. These have, however, generally not been tested in cheese applications, nor has their substrate specificity been tested towards milk fat. The pregastric esterases are expected to be similar to those of the ruminants. The other, non-pregastric, lipases range in specificity from long chain fatty acids in lipids to true esterases, like the pig liver esterase.